Hybrid Lipid/Polymer Nanoparticles
for Pulmonary Delivery of siRNA:
Development and Fate Upon In Vitro Deposition
on the Human Epithelial Airway Barrier
Ivana d’Angelo, PhD,1Gabriella Costabile, PhD,2,3Estelle Durantie, PhD,3 Paola Brocca, PhD,4Valeria Rondelli, PhD,4Annapina Russo, PhD,5 Giulia Russo, PhD,5
Agnese Miro, PharmD,2Fabiana Quaglia, PhD,2Alke Petri-Fink, PhD,3 Barbara Rothen-Rutishauser, PhD,3and Francesca Ungaro, PhD2
Abstract
Background: Nowadays, the downregulation of genes involved in the pathogenesis of severe lung diseases
through local siRNA delivery appears an interesting therapeutic approach. In this study, we propose novel
hybrid lipid-polymer nanoparticles (hNPs) consisting of poly(lactic-co-glycolic) acid (PLGA) and dipalmitoyl
phosphatidylcholine (DPPC) as siRNA inhalation system.
Methods: A panel of DPPC/PLGA hNPs was prepared by emulsion/solvent diffusion and fully characterized. A
combination of model siRNAs against the sodium transepithelial channel (ENaC) was entrapped in optimized
hNPs comprising or not poly(ethylenimine) (PEI) as third component. siRNA-loaded hNPs were characterized for
encapsulation efficiency, release kinetics, aerodynamic properties, and stability in artificial mucus (AM). The fate
and cytotoxicity of hNPs upon aerosolization on a triple cell co-culture model (TCCC) mimicking human epithelial
airway barrier were assessed. Finally, the effect of siRNA-loaded hNPs on ENaC protein expression at 72 hours
was evaluated in A549 cells.
Results: Optimized muco-inert hNPs encapsulating model siRNA with high efficiency were produced. The
de-veloped hNPs displayed a hydrodynamic diameter of *150 nm, a low polydispersity index, a negative f potential
close to
-25 mV, and a peculiar triphasic siRNA release lasting for 5 days, which slowed down in the presence of PEI.
siRNA formulations showed optimal in vitro aerosol performance after delivery with a vibrating mesh nebulizer.
Furthermore, small-angle X-ray scattering analyses highlighted an excellent stability upon incubation with AM,
confirming the potential of hNPs for direct aerosolization on mucus-lined airways. Studies in TCCC confirmed that
fluorescent hNPs are internalized inside airway epithelial cells and do not exert any cytotoxic or acute
proin-flammatory effect. Finally, a prolonged inhibition of ENaC protein expression was observed in A549 cells upon
treatment with siRNA-loaded hNPs.
Conclusions: Results demonstrate the great potential of hNPs as carriers for pulmonary delivery of siRNA,
prompting toward investigation of their therapeutic effectiveness in severe lung diseases.
Keywords:
dipalmitoylphosphatidylcholine, inhalable nanoparticles, poly(ethylenimine), poly(lactic-co-glycolic)
acid, siRNA, triple cell coculture
1Di.S.T.A.Bi.F., University of Campania ‘‘Luigi Vanvitelli,’’ Caserta, Italy.
2
Laboratory of Drug Delivery, Department of Pharmacy, University of Napoli Federico II, Napoli, Italy.
3
Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland.
4
Applied Physics, Department of Medical Biotechnology and Translational Medicine, University of Milano, Milano, Italy.
5
Laboratory of Biochemistry, Department of Pharmacy, University of Napoli Federico II, Napoli, Italy. ª Mary Ann Liebert, Inc.
Pp. 170–181
DOI: 10.1089/jamp.2017.1364
170
Introduction
R
NA interferencerefers to the inhibition of gene ex-pression by small, double-stranded RNA molecules (small interference RNA or siRNA) that direct the mRNA machinery to degrade a specific mRNA. siRNA therapeutics have many advantages in terms of clinical applications, the first of which is their virtual potential to specifically silence any gene underlying more-or-less complex pathologies.(1,2) In particular, severe lung diseases, such as lung cancer or cystic fibrosis (CF), nowadays represent a very important area of application for siRNA-based therapies.(3) Never-theless, to harness the full potential of the siRNA strategy for severe lung diseases, an appropriately designed delivery system is mandatory.(4,5)Naked siRNAs have a half-life of less than an hour in human plasma and are often unable to penetrate cell membranes. In fact, cellular uptake and the escape from endolysosomes are critical steps before siRNA reaches the cytoplasm, where its target is located. Conceiving siRNA for local delivery by in-halation, the presence of high mucin, DNA, and actin con-centrations, makes airway mucus a very complex barrier to overcome.(4,5) Thus, optimal inhalable formulations are re-quired: (i) to enhance siRNA stability; (ii) to overcome lung cellular and noncellular barriers; and (iii) to increase siRNA availability at the target level.
Amid advanced drug delivery systems, the encapsulation of siRNA into colloidal carriers is considered a very promising formulation approach for inhaled treatment of severe lung dis-eases.(4,5)In this context, biodegradable poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) are gaining considerable in-terest since they may provide protection of the therapeutic cargo from enzymatic degradation, prolonged release (i.e., decreased number of administrations), and improved retention in the lungs.(6,7) Nevertheless, particle engineering with excipients aimed to overcome the mucus-lined human lung epithelial barrier is essential.(8,9)
In the attempt to combine the most valuable properties of both lipid and polymeric nanocarriers, hybrid lipid-polymer NPs (hNPs) have recently been proposed.(10,11) Thanks to their ability to form a shell upon the polymer core, lipids may be useful to impart peculiar surface properties to the carrier, likely tuning NP interactions with the lung envi-ronment, while enhancing their tolerance in the pulmonary tract.(10)In fact, the use of endogenous phospholipids, such as dipalmitoylphosphatidylcholine (DPPC), can be consid-ered a valid approach to increase NP compatibility with the lung environment. Furthermore, DPPC has been shown to improve the technological features of PLGA particles aimed to nucleic acid delivery to the lung.(12,13)
Along these lines, the aim of this work was to design and optimize hNPs consisting of PLGA and DPPC, introducing PEI as a third component to assist siRNA encapsulation in the hNPs. A preliminary formulation study allowed to select op-timized muco-inert DPPC/PLGA and PEI/DPPC/PLGA hNPs displaying optimal in vitro aerosol performance. A particular attention was devoted to the characterization of the carrier upon contact with simulated lung fluids through a combination of analytical techniques, that is spectrophotometric assay, dynamic light scattering (DLS), and small-angle X-ray scattering (SAXS). To allow a proof of principle of RNP potential for siRNA delivery to the lung, selected formulations were loaded with
model therapeutic siRNAs of interest for CF therapy, which is a combination of siRNA against the a and b subunits of the sodium transepithelial channel (ENaC). siRNA-loaded hNPs were fully characterized for size, zeta potential, en-capsulation efficiency, and in vitro release kinetics. A thorough in vitro study allowed to assess the fate and the effect of hNPs upon aerosolization on a triple cell co-culture model (TCCC) grown at air–liquid interface (ALI) mim-icking the human airway epithelial barrier.(14) Finally, the in vitro effects of siRNA-loaded hNPs on the expression of aENaC and bENaC subunits were evaluated on a model human lung epithelial cell line.
Materials and Methods Materials
ResomerRG 502H (uncapped PLGA 50:50, inherent vis-cosity 0.16 0.24 dL/g) was purchased from Evonik Industries AG (Germany). Rhodamine-labeled PLGA was synthesized as previously described.(15) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was a kind gift of Lipoid GmbH (Switzerland). siGENOME SMARTpool siRNA against a and b subunits of the sodium transepithelial channel (ENaC) (Human SCNN1A and SCNN1B; 19 mer duplexes; Mw= 13.4 kDa) and siGENOME Non-Targeting siRNA Pool were purchased from Dharmacon (GE Healthcare).
Ph.Eur. grade mannitol (Pearlitol C160) was kindly gifted by Roquette Italia S.p.a. (Italy). Egg yolk emulsion, deoxyribonucleic acid sodium salt from calf thymus (DNA), diethylenetriaminepentaacetic acid (DTPA), phosphate buffer salts, branched polyethylenimine (PEI; 25 kDa), potassium chloride, rhodamine B (Rhod-B), RPMI 1640 amino acid solution, sodium chloride, and mucin from porcine stomach were purchased from Sigma-Aldrich (Missouri). Methylene chloride and ethanol 96% (v/v) were supplied by Carlo Erba (Italy).
Production of DPPC/PLGA hybrid NPs
DPPC/PLGA hNPs were prepared by an emulsion/solvent diffusion technique. Briefly, a water in oil emulsion (w/o) was achieved adding 100 lL of water to 1 mL of methylene chlo-ride containing PLGA (10 mg; 1% w/v) and DPPC at different DPPC/PLGA weight ratios (1:10, 1:20, 1:50, 1:100, and 1:150), under vortex mixing (2400 minutes-1; Heidolph, Ger-many). When needed, PEI was added to the internal water phase at the theoretical loading of 0.016 mg per 100 mg of PLGA. Just after mixing, the w/o emulsion was added to 12.5 mL of ethanol 96% (v/v) under moderate magnetic stir-ring, leading to immediate nanoparticle precipitation. Then, the formulation was diluted with 12.5 mL of Milli-Q water under stirring for 10 minutes. Afterward, residue organic solvent was removed by rotary evaporation under vacuum at 30C (Hei-dolph VV 2000, Germany). hNPs were isolated from the re-sulting hNP colloidal dispersion (5 mL) by centrifugation at 7000 rcf for 20 minutes at 4C (Hettich Zentrifugen, Germany) and dispersed in Milli-Q water.
siRNA-loaded hNPs were prepared in optimized formu-lation conditions at a theoretical loading of 1 nmol/100 mg of PLGA (N/P theoretical ratio of 10 in PEI-modified hNPs) by adding siRNA to the internal water phase.
Fluorescently-labeled hNPs (DPPC/PLGARhodand DPPC/
PLGARhod) were prepared using rhodamine-labeled PLGA
(PLGA-Rhod) in the organic phase at 10% w/w with respect to the total PLGA amount.
Characterization of DPPC/PLGA hybrid NPs
Size and zeta potential. The hydrodynamic diameter
(DH), the polydispersity index (PI), and the zeta potential (f
potential) of hNPs were determined by DLS, with a Malvern Zetasizer Nano ZS (Malvern Instruments, United King-dom). Results are expressed as mean value– standard de-viation (SD) of triplicate measurements in different batches.
siRNA loading inside hNPs. siRNA actual loading was
measured indirectly by quantifying the amount of un-encapsulated siRNA. Briefly, just after production, hNPs were collected by centrifugation (7000 rcf for 20 minutes at 4C) and the supernatant was analyzed for siRNA content using Quant-IT RiboGreenreagent (Thermo Fisher Sci-entific, Massachusetts, Stati Uniti) according to the manufac-turer’s instructions. Quantitative analysis was performed by spectrofluorimetry at kex 480 nm/kem 520 nm (EnVision
Multilabel Plate Readers, PerkinElmer, Inc.). Results are reported as actual loading (nmol of encapsulated siRNA per mg of yielded hNPs) and encapsulation efficiency (actual loading/theoretical loading· 100) – SD of values collected from three different batches.
SAXS spectroscopy. Synchrotron SAXS measurements
were performed at the ID02 high-brilliance beamline(16)at the ESRF (Grenoble, France), with a beam cross section of 200· 400 lm and wavelength k = 0.1 nm, using two different sample detection distances: 1 and 6 m. The range of investi-gated momentum transfer, q= (4p/k)sin(h), was 0.0116 nm-1 <q <6.43 nm-1, where 2 h is the scattering angle. All mea-surements were performed at T= 25C. Samples were put in plastic capillaries (KI-BEAM, ENKI srl) with 2 mm internal diameter, 0.05 mm wall thickness, and closed with polyeth-ylene caps. Capillaries were then mounted horizontally onto the sample holder. Sample concentration was 10 mg/mL. The exposure time of each measurement was 0.1 seconds, and spectra were checked for radiation damage.
The measured SAXS profiles report the scattered radia-tion intensity as a funcradia-tion of the momentum transfer, q, where 1 m and 6 m sample-to-detector distance spectra were merged. Solvent subtraction was obtained by measuring water-filled capillaries and empty capillaries.
In vitro interaction with mucin. The light scattering of hNP
aqueous dispersions with or without mucin was assessed by spectrophotometry as previously described.(8,17) Briefly, a saturated solution of type II mucin was prepared by centri-fugation at 6000 rcf for 20 minutes in a mucin dispersion in water (0.08% w/v) stirred overnight. Then, hNPs were dis-persed in the mucin solution at a concentration of 1 mg/mL by vortexing for 1 minute. The light scattering of the mucin/ hNPs mixtures was measured by spectrophotometric analysis at 650 nm at time 0 and after incubation for 30 and 60 minutes at room temperature. Reference absorbances of mucin and 1 mg/mL hNP dispersions in water were also evaluated.
Experiments were run in triplicate and results are expressed as absorbance at 650 nm– SD over time.
The interactions of hNPs with mucin were further inves-tigated by DLS as previously reported.(9) Briefly, the hNP dispersions in mucin were examined with the Malvern Zeta-sizer Nano ZS (Malvern Instruments, United Kingdom). Experiments were run in triplicate and results are reported as representative size distribution by intensity of hNPs in mucin versus hNPs in water. The size distribution profile of the mucin solution was also recorded as control.
Particle stability in artificial mucus. To assess the stability
of siRNA-loaded hNPs and their ability to diffuse inside airway mucus, SAXS analyses were performed on hNP dis-persions in the presence of artificial mucus (AM). As previ-ously reported,(8)AM was prepared by adding 25 lL of sterile egg yolk emulsion, 25 mg of mucin, 20 mg of DNA, 30 lL of aqueous DTPA (1 mg/mL), 25 mg NaCl, 11 mg KCl, and 100 lL of RPMI 1640 to 5 mL of water. The dispersion was stirred until a homogenous mixture was obtained. For stability studies, aqueous dispersions of siRNA-loaded hNPs (10 mg/ mL) were mixed with AM in a 1:3 volume ratio for a total 40 lL sample volume. After a 1-hour equilibration time, SAXS was performed as described above. Particle structure after a 12-hour incubation with AM was verified by repeated SAXS measurements.
In vitro release kinetics of siRNA. siRNA-loaded hNPs
were characterized for in vitro siRNA release kinetics in phosphate buffer (120 mM NaCl, 2.7 mM KCl, and 10 mM Na2HPO4) at pH 7.2 (phosphate-buffered saline [PBS]). Release studies were performed upon dilution of hNP dis-persions in PBS to a theoretical siRNA concentration of 0.2 nmol/mL. hNP dispersions were incubated in a horizontal shaking water bath at 37C (ShakeTemp SW 22, Julabo, Italy). At scheduled time intervals, samples were centrifuged at 7000 rcf for 20 minutes at 4C to isolate hNPs and the release medium was withdrawn and analyzed for siRNA content by spectrofluorimetric analysis as described for actual loading. The medium was replaced by the same amount of fresh PBS. Experiments were carried out in triplicate and results expressed as the percentage of siRNA released– SD.
In Vitro aerosol performance. The aerosolization
prop-erties of fluorescently-labeled DPPC/PLGARhod and PEI/
DPPC/PLGARhodhNP dispersions were investigated in vitro
after delivery from the AeronebGo nebulizer (Aerogen Ltd., Ireland) by a Next Generation Impactor (NGI) (Copley Sci-entific, United Kingdom) according to the Comite´ Europe´en de Normalization (CEN) standard methodology for nebulizer systems with sampling at 15 L/minutes and insertion of a filter in micro-orifice collector (MOC).(18) Briefly, 1 mL of hNP dispersion was transferred to the reservoir of the nebulizer, which was connected to the induction port of the NGI. The nebulizer was operated at 15 L/minutes, and the aerosol was drawn through the impactor for 5 minutes, until dryness.
The amount of fluorescent hNPs remaining inside the nebulizer chamber, deposited on the seven NGI collection cups, and in the induction port was quantitatively recovered in NaOH 0.5 M. After stirring at room temperature for 1 hour, the amount of fluorescent hNPs in the resulting so-lutions was determined by spectrofluorimetric analysis at kex
553 nm/kem577 nm (GloMaxExplorer, Promega, Italy) as
previously reported.(8) Calibration curves were derived by analyzing serial dilutions of DPPC/PLGARhodor PEI/DPPC/
PLGARhodstandard solutions prepared from a stock of hNPs
degraded in NaOH 0.5 M. The linearity of the response was verified over the concentration range 2–200 lg/mL (r2 ‡ 0.999). Each experiment was run in triplicate.
The emitted dose (ED) was measured as the difference be-tween the total amount of hNPs initially placed and the amount remaining in the nebulizer chamber. Upon emission, the ex-perimental mass median aerodynamic diameter (MMADexp)
and the geometric SD (GSD) were calculated according to the European Pharmacopoeia, deriving a plot of cumulative mass of particle retained in each collection cup (expressed as percent of total mass recovered in the impactor) versus the cutoff di-ameter of the respective stage.
The fine particle fraction (FPF) was calculated taking into account the amount of hNPs deposited in stages 3–7 (MMA-Dexp<5.39 lm) compared to the initial amount loaded into the
nebulizer chamber, while the respirable fraction (RF) referred to the total amount recovered from the NGI.
In vitro studies on airway triple cell cocultures
All in vitro exposure experiments were conducted with a three-dimensional TCCC mimicking the human epithelial airway barrier comprising human bronchial epithelial cells (16HBE14o-), human blood monocyte-derived macrophages (MDM), and dendritic cells (MDDC).(14)
16HBE14o- cell cultures. 16HBE14o- cells were kindly
provided by Dieter Gruenert (passage number P2.54; Uni-versity California, San Francisco, CA). Cells were maintained in minimum essential media 1· (with Earle’s Salts, 25 mM HEPES and without l-glutamine; Gibco BRL, Thermo Fisher Scientific), supplemented with 1% l-glutamine, 1% penicil-lin/streptomycin, and 10% fetal calf serum.
For experimental cultures, cells were seeded at a density of 0.5· 106cells/insert on transparent BD Falcon (Corning)
cell culture inserts (surface area of 0.9 cm2, pores with 3.0 lm diameter, and PET membranes for 12-well plates). The cell culture inserts were pretreated with 150 lL of a fibronectin coating solution containing 0.1 mg/mL bovine serum albumin (Sigma-Aldrich), 1% Type I bovine collagen (BD Biosciences, Becton Dickinson, New Jersey), and 1% human fibronectin (BD Biosciences, Becton Dickinson) in the LHC Basal Medium (Sigma-Aldrich). Inserts were placed in BD Falcon tissue culture plates (12-well plates) with 0.5 mL medium in the upper and 1.5 mL in the lower chamber. The cells were kept at 37C in 5% CO2humidified
atmosphere for 7 days (medium changed after 3–4 days).
TCCC system. To produce the TCCC in vitro model,
16HBE14o- cell cultures were supplemented on the apical side with MDM and on the basolateral side with MDDC derived from human blood as previously reported.(14,19) Briefly, 16HBE14o- cells were cultured as described above. At 5 days, the medium was removed from the upper and lower chamber, the inserts turned upside down, and the bottom was abraded carefully with a cell scraper. The inserts were then incubated with 65 lL medium containing 83· 104 MDDC/mL on the basal side for 2 hours. Afterward, the inserts were turned
around again and placed in BD Falcon (Corning) tissue culture plates (12-well plates). In the lower chamber, 1.5 mL of me-dium, and at the apical side of the TCCC, 500 lL medium containing 2.5· 104
MDM/mL were added. The complete TCCC systems were kept with 1.5 mL of medium in the lower chamber and 0.5 mL in the upper chamber at 37C in 5% CO2
humidified atmosphere for 24 hours.
To establish the ALI, the medium in the upper chamber of the TCCC systems was removed 24 hours before exposure in the Vitrocell Cloud, while the medium in the lower chamber was replaced by 0.6 mL of fresh medium.
In vitro TCCC exposure to aerosolized NPs. Cell
expo-sure to hNPs at ALI was carried out with the Vitrocell Cloud system (Vitrocell Systems GmbH, Germany). Briefly, the Vitrocell Cloud consists of three main components: a nebu-lizer, an exposure chamber, and a flow system, with an in-cubation chamber providing suitable temperature and humidity cell culture conditions. The aerosol is generated into the exposure chamber by a perforated vibrating membrane nebulizer (AeronebPro, with a span of 2.5–6.0 lm volume mean diameter). Before each experiment, freeze-dried hNPs were dispersed in 0.5 mM sodium chloride.
For TCCC exposure to low hNP concentrations, 200 lL of a 0.5 mg/mL hNP dispersion was aerosolized (0.1 mg of hNPs). For high hNP concentrations, TCCC were exposed to further 400 lL (2· 200 lL) of a 0.9 mg/mL hNP dispersion (0.5 mg of hNPs). TCCC exposed to 0.5 mM sodium chloride and mannitol aqueous solutions was used as controls. Fol-lowing the exposure in the chamber (10 and 30 minutes for 0.1 and 0.5 mg of hNPs, respectively), the inserts were im-mediately placed in new BD Falcon 12- well tissue culture plates containing 0.6 mL of fresh medium for cell uptake studies, cytotoxicity assay, and tumor necrosis factor alpha (TNF-a) release. The morphology of hNPs was analyzed by transmission electron microscopy (TEM) before nebulization and after exposing a 400 mesh copper film grid to nebulized hNPs. The analysis was performed with a FEI Tecnai spirit TEM at 120 kV and images were acquired with a Veleta CCD camera (2048· 2048 pixels).
Nanoparticle deposition efficiency. The amount of hNPs
deposited upon nebulization inside the Vitrocell Cloud was assessed. To this purpose, glass coverslips (24· 24 mm) were placed onto the compartments for cell culture inserts and ex-posed to different amounts of aerosolized rhodamine-labeled DPPC/PLGARhodand PEI/DPPC/PLGARhodhNPs. After the
exposure, coverslips were removed and rinsed with NaOH 0.5 M (500 lL). After stirring at room temperature for 1 hour, the amount of fluorescent hNPs in the solutions was quantified by spectrofluorimetric analysis as described for in vitro aerosol performance. Results are reported as lg of hNPs deposited per cm2– SD of values collected from triplicate analyses.
Cell uptake studies. The TCCC was exposed to different
amounts of aerosolized rhodamine-labeled DPPC/PLGARhod
and PEI/DPPC/PLGARhod hNPs. After 24 hours
postexpo-sure, TCCC were fixed for 15 minutes with 3% paraformal-dehyde in PBS at room temperature and cells were washed thrice in PBS. The F-actin cytoskeleton and the DNA of all cells were stained with phalloidin (ALEXA 488) (Molecular Probes, Life Technologies Europe B.V., Switzerland) and
4¢,6- diamidin-2-phenylindol (DAPI) (1 lg/mL) in a 1:100 and 1:50 dilution in 0.3% Triton X-100 in PBS, respectively. For microscopy, membranes were embedded in Glycergel (DAKO Schweiz AG, Baar, Switzerland). Sample analysis was performed with an inverted Zeiss confocal laser scanning microscope 710 (LSM, Axio Observer.Z1, Zeiss, Germany). Representative images (z-stacks) were recorded at three in-dependent fields of view for each sample. Image processing was achieved using the 3D reconstruction software Imaris (Bitplane AG, Switzerland).
Cytotoxicity assay. The TCCC was exposed to different
amounts of aerosolized siRNA-DPPC/PLGA and siRNA-PEI/ DPPC/PLGA, and corresponding empty hNPs. After 24 hours postexposure at 37C, lactate dehydrogenase (LDH) release by the TCCC, indicative of cell membrane damage, was as-sessed by the LDH cytotoxicity detection kit (Roche Applied Science, Germany) according to the manufacturer’s guide-lines. The test was performed in triplicates and evaluated in comparison to the negative controls, which are TCCC ex-posed to aqueous solutions of 0.5 mM sodium chloride or mannitol at the same concentrations of hNP dispersions. As a positive control, TCCC were treated with 100 lL of 0.2% Triton X-100 in PBS on the apical side and incubated for 24 hours at 37C and 5% CO2.
Tumor necrosis factor-a release. The potential
proin-flammatory response was also investigated after 24 hours postexposure to hNPs by quantifying the release of TNF-a using an ELISA kit (R&D Systems) according to the man-ufacturer’s protocol.
In vitro gene silencing effect
Cell cytotoxicity. A549 cells were seeded onto 96-well
plates (5· 104cells/well) and incubated with hNPs for 24
and 48–72 hours. Then, cell viability was evaluated by MTT assay as previously reported.(20a)
Cell culture, transfection, and treatment. Human alveolar
basal epithelial cell line (A549) was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum ( Microtech), 1.5 mM L-glutamine, 100 units/mL penicillin, and 100 lg/mL streptomycin
under humidified atmosphere of 5% CO2at 37C. siRNA
transfections and treatments were performed as previously described.(20b)
Western blotting. Total proteins (75 lg) were extracted
from the cells, separated on 10% sodium dodecyl sulfate– polyacrylamide gel electrophoresis and electrotransferred on polyvinylidene fluoride (PVDF) as previously described.(21) Then, the membrane was incubated for 2 hours with 5% nonfat milk to block nonspecific binding sites and incubated 2 hours at room temperature with the following bodies: bENaC (Santa Cruz Biotechnology), anti-SCNN1A (Proteintech Europe), and anti-b-actin (Santa Cruz Biotechnology). Densitometric analysis was carried out using ImageJ software (Image 1.49v, available on: http://imagej. nih.gov/ij, National Institutes of Health, Bethesda, MD).
Data analysis
Cell studies were performed in triplicate and results are expressed as mean value– standard error mean (n = 3). The significance of differences was determined with the software Kaleida graph using one-way analysis of variance followed by Bonferroni post-hoc test. A p-value<0.05 was considered significant.
Results
Development studies of DPPC/PLGA hNPs for siRNA loading
In-depth formulation studies were performed on empty hNPs to establish the optimal ratio between the selected components, that is, PLGA, DPPC, and PEI. Overall hNP properties are reported in Table 1.
Independent of the amount of DPPC added to the organic phase and the presence of PEI in the internal water phase, the adopted formulation conditions lead to the formation of hNPs with DHranging between *135 and *169 nm, low PI
(<0.130), and negative f potentials (between -16 and -30 mV). Nevertheless, in each case, a flocculation of the exceeding amount of the lipid component was apparent after rotary evaporation of the diffusion solvent under vacuum.
This phenomenon was particularly evident when high amounts of DPPC were added to the formulation (i.e., DPPC/
Table1. Composition, Size, Polydispersity Index, and Zeta Potential of Empty DPPC/PLGA and PEI/DPPC/PLGA Hybrid Lipid-Polymer Nanoparticles
Formulation PEI theoretical loading (mg/100 mg of PLGA) DPPC/PLGA ratio (w/w) DH (nm– SD) PI (mean– SD) f potential(mV– SD) DPPC/PLGA10 — 1:10 151.3– 21.3 0.127– 0.079 -25.3 – 0.5 DPPC/PLGA 20 — 1:20 149.7– 2.1 0.083– 0.006 -23.2 – 2.4 DPPC/PLGA 50 — 1:50 135.4– 17.2 0.088– 0.008 -24.3 – 6.0 DPPC/PLGA 100 — 1:100 158.9– 7.1 0.082– 0.004 -28.4 – 2.9 DPPC/PLGA 150 — 1:150 168.6– 7.1 0.124– 0.094 -15.7 – 1.3 PEI/DPPC/PLGA 10 0.016 1:10 166.5– 13.9 0.128– 0.093 -26.1 – 0.5 PEI/DPPC/PLGA 20 0.016 1:20 165.4– 9.1 0.102– 0.006 -31.7 – 1.2 PEI/DPPC/PLGA 50 0.016 1:50 162.3– 13.9 0.124– 0.041 -28.0 – 0.7 PEI/DPPC/PLGA 100 0.016 1:100 150.9– 30.4 0.106– 0.027 -28.0 – 0.3 PEI/DPPC/PLGA 150 0.016 1:150 146.4– 0.3 0.123– 0.027 -22.2 – 2.7
PI, polydispersity index; SD, standard deviation.
PLGA 1:10 w/w), but it was limited up to a theoretical DPPC/ PLGA ratio of 1:20. Thus, this last ratio was selected for further studies to maximize the amount of DPPC associated to the NPs, while limiting flocculation. The corresponding formulations, from here on, are named DPPC/PLGA and PEI/DPPC/PLGA. Mucin/nanoparticle interaction studies were performed by measuring the absorbance at 650 nm of DPPC/PLGA and PEI/DPPC/PLGA hNPs in mucin over time (Fig. 1A). While
the absorbance of the reference mucin dispersion was close to zero, higher values of absorbance were observed at each time point upon addition of hNP dispersions. Nevertheless, no significant differences were observed between absorbance of hNPs dispersed in water or mucin (Fig. 1A). The interactions between mucin and either DPPC/PLGA or PEI/DPPC/PLGA hNP were analyzed also by DLS (Fig. 1B, C, respectively).
The combined particle size distribution for the various replicates shows no change in the position and intensity of the peak. In line with previous studies,(9)the apparent dimension of bare PLGA is significantly shifted to larger sizes upon addition of mucin, reflecting strong mucin/NP hydrophobic interac-tions, which likely provide a basis for mucoadhesive NPs (Supplementary Fig. S1A; Supplementary Data are available online at www.liebertpub.com/jamp). A similar behavior was observed for bare PEI-PLGA NPs (Supplementary Fig. S1B).
Characterization of siRNA-loaded DPPC/PLGA hNPs
A model siRNA against ENaC was entrapped in DPPC/ PLGA and PEI/DPPC/PLGA formulations at the theoretical loading of 1 nmol/100 mg of PLGA. The addition of siRNA inside the formulation did not result in significant changes of hNP size and PI (Table 2) compared to empty hNPs (Table 1). siRNA-loaded hNPs with a DHaround 140 nm (141 and 142 nm
for siRNA-DPPC/PLGA and siRNA-PEI/DPPC/PLGA, re-spectively) and low PI were produced with good yields (52% and 53% for siRNA-DPPC/PLGA and siRNA-PEI/DPPC/ PLGA, respectively). siRNA was effectively incorporated in-side DPPC/PLGA hNPs with a mean encapsulation efficiency of 75%. The addition of PEI inside the formulation allowed to reach an excellent encapsulation efficiency of 96%, thus increasing siRNA actual loading from 1.35 to 1.74 nmol per 100 mg of hNPs.
Results of in vitro release studies performed at physio-logical pH and temperature (pH 7.2 and 37C) are reported in Figure 2 as percentage of siRNA released over time.
Both siRNA-DPPC/PLGA and siRNA-PEI/DPPC/PLGA hNPs displayed a typical triphasic release profile, characterized by an initial burst, with more than 50% of siRNA released in the first hours, followed by a slow release phase lasting a couple of days and a final fast release time period after 4–5 days. Al-though, both formulations show similar release profile, siRNA/ FIG. 1. In vitro assessment of mucin interactions with
op-timized empty DPPC/PLGA and PEI/DPPC/PLGA hNPs. (A) Scattering at 650 nm of hNPs in mucin (1 mg/mL) over time. The absorbance at 650 nm of mucin and hNP dispersions in water is reported as control. (B–C) Size distribution by inten-sity of hNP dispersions in water or a saturated mucin solution evaluated by DLS. The size distribution profile of mucin in water is reported as control (B DPPC/PLGA hNPs and C PEI/ DPPC/PLGA). hNPs, hybrid lipid-polymer nanoparticles. Color images available online at www.liebertpub.com/jamp
Table 2. Properties of siRNA-Loaded Hybrid Lipid-Polymer Nanoparticles siRNA-DPPC/ PLGA siRNA-PEI/ DPPC/PLGA DH(nm– SD) 141– 0.50 142– 1.8 PI (mean– SD) 0.120– 0.018 0.114– 0.0050 f potential (mV– SD) -29.1 – 1.6 -29.9 – 1.4 Yield of production (%– SD) 52.0– 8.9 53.4– 2.5 siRNA actual loadinga
(%– SD)
1.35– 0.02 1.74– 0.04 Encapsulation efficiency
(%– SD) 74.8– 1.5 95.9– 2.2
a
siRNA loading was expressed as nmol of encapsulated siRNA per 100 mg of hNPs. siRNA theoretical loading was 1 nmol/100 mg of hNPs.
hNPs, hybrid lipid-polymer nanoparticles.
DPPC/PLGA hNPs provided a significantly more rapid release compared to siRNA-PEI/DPPC/PLGA hNPs. Nevertheless, both hNP formulations sustained siRNA release for 1 week.
The I(q) SAXS spectra of DPPC/PLGA and siRNA-PEI/DPPC/PLGA hNPs are reported in Figure 3 (Top panel). A low q region affected by the finite size of the particle in the range of the 100 nm distances can be identified. At higher q, an extended region with q-4 slope shows up, which identifies a sharp solvent–particle interface and a smooth particle surface. Giunier analysis(22)in the interval 0.0116 nm-1<q <0.0246 nm-1 gives gyration radii Rg= 53 – 0.5 nm and Rg = 52 – 0.6 nm for siRNA-DPPC/PLGA and siRNA-PEI/DPPC/PLGA hNPs, respectively. If modeled as spheres, this corresponds to particle radii of 68 and 67 nm, in good agreement with hydrodynamic radii (Table 2). The best fit of I(q) is obtained by applying the form factor of a distribution of polydispersed spheres with a number-averaged mean radius of 45– 9 nm for siRNA-DPPC/ PLGA and 42– 11 nm for siRNA-PEI/DPPC/PLGA hNPs. The fits are shown in Figure 3 (red lines).
The phospholipid component is possibly coating the PLGA particle, without forming a bilayer envelope. A comparison of the spectra of bare PLGA NPs and DPPC/PLGA hNPs is reported in Supplementary Figure S2 to better illustrate this point. We underline that PEI and siRNA contents are actually of the order of lg over 10 mg of particles, so that scattering contribution from those components are not detectable.
Applying SAXS analysis, the stability of hNPs in mucus was studied during 12 hours after mixing AM with aqueous dispersions of hNPs in a 3:1 volume ratio. The scattering spectra obtained after equilibration over 12 hours are reported in Figure 3 (Bottom panel). Of note, the obtained spectrum is consistent with the sum of the naked particle spectrum and the mucus spectrum that give the reconstructed line shown in the figure (blue line).
In vitro aerosol performance of the optimized hNP for-mulations was first assessed through NGI. To easily quantify
the amount of hNPs deposited in each cup, blank fluorescent DPPC/PLGARhodand PEI/DPPC/PLGARhodhNPs were used
(Fig. 4). In fact, neither the presence of siRNA nor the addi-tion of PLGA-Rhod affected hNP size and surface properties, which are crucial for aerosolization.
According to the new regulatory guidance, both USP and Ph.Eur. recognize the suitability of the NGI for nebulizer characterization when used at 15 L/min excluding the pre-separator and placing an internal filter below the MOC.(18)In this configuration, the seven NGI stages produce cutoff diam-eters in the range 14.1–0.98 lm, while the last five stages have cutoff diameters between 5.39 and 0.98 lm. Figure 4A de-scribes the cumulative droplet size distribution of the hNP dose emitted after aerosolization from an Aeroneb Go vibrating-mesh nebulizer. In both cases, the best fit line was from loga-rithmic linear regression (r2> 0.96). The deposition patterns of DPPC/PLGARhodand PEI/DPPC/PLGARhodthroughout the
NGI cups are shown in Figure 4B. In both cases, less than 1.5% of the ED of hNPs was deposited in the throat, with 65%–75% of the ED recovered from Cups 3–7 (MMADexp<5.39 lm).
FIG. 2. In vitro release kinetics of siRNA from hNPs in PBS at pH 7.2. Data are expressed as siRNA released (%)– SD (n= 3). PBS, phosphate-buffered saline; SD, standard devia-tion. Color images available online at www.liebertpub.com/ jamp
FIG. 3. Small-angle X-ray scattering spectra of siRNA-loaded hNPs. Top panel: siRNA-DPPC/PLGA (full dots) and siRNA-PEI/DPPC/PLGA (empty dots) in water. The red line is the representative fitting of the siRNA-PEI/ DPPC/PLGA data with the form factor of a polydisperse sphere (red line). Bottom panel: siRNA-DPPC/PLGA (top) and siRNA-PEI/DPPC/PLGA (bottom) after 12 hours of incubation with artificial mucus (AM) (empty dots) versus a reconstructed spectrum obtained by summing up AM and naked hNP spectra (blue line). siRNA-DPPC/PLGA spec-trum is stacked for clarity. Color images available online at www.liebertpub.com/jamp
Nevertheless, while DPPC/PLGARhod formulation showed a
peak at 5.39 lm, the highest percent of PEI/DPPC/PLGARhod
was recovered from cup with a cutoff diameter of 3.3 lm. As a consequence, by the same value of GSD, PEI/DPPC/PLGARhod
hNPs displayed a lower MMADexpand, consequently, higher
FPF and RF values compared to DPPC/PLGARhod(Fig. 4C).
In vitro aerosolization of hNPs on TCCC
The ability of aerosolized hNPs to penetrate airway epi-thelial barrier was probed in vitro on TCCC. The experiments were carried out on fresh dispersions of freeze-dried hNPs (1:25 hNP/mannitol weight ratio) in 0.5 mM NaCl for ideal nebulization. Mannitol was added to the hNPs as cryopro-tectant to avoid common hNP aggregation in a collapsed cake.(23)In fact, it induced the lowest variation of DHand PI
with respect to the control, and no difference was observed when changing the hNP/cryoprotectant ratio (Supplementary Table S1 and Supplementary Fig. S3). Furthermore, mannitol could also work facilitating hNP transport through mucus, and in so doing siRNA diffusion, due to its established ability to improve fluidity of mucus.(24)
The stability of hNPs during the nebulization process was confirmed by transmission electron microscopy (TEM) analysis of freeze-dried siRNA-loaded hNPs in the presence of mannitol (1:25 NP/mannitol ratio) before and after neb-ulization in the Vitrocell Cloud (Fig. 5). TEM images sug-gest a potential core-shell structure for both siRNA-DPPC/ PLGA and siRNA-PEI/DPPC/PLGA, which was retained after the nebulization process, which did not cause any ap-parent modification of the hNP structure or hNP aggregation for each tested concentration.
The deposition efficiency was also evaluated for both tested formulations, highlighting in each case a homogeneous dis-tribution of the hNPs on the coverslips inside the exposure chamber (Supplementary Fig. S4) and a direct correlation be-tween the delivered and deposited doses of hNPs. In particular, 0.0051– 0.0018 and 0.0127 – 0.0058 lg/cm2 of hNPs were
deposited on coverslips after nebulization of DPPC/PLGARhod
dispersions at low and high concentrations, respectively.
FIG. 4. In vitro aerosol performance of DPPC/PLGA and PEI/DPPC/PLGA hNPs upon delivery through the Aero-neb Go nebulizer. (A) Cumulative mass recovered as a function of the cutoff diameter of the NGI stages; (B) NGI deposition pattern; (C) fine particle characteristics of the aerosol cloud. Data are presented as mean– SD (n = 3). NGI, Next Generation Impactor. Color images available online at www.liebertpub.com/jamp
FIG. 5. Transmission electron microscopy (TEM) images of reconstituted freeze-dried siRNA-loaded hNPs (siRNA-DPPC/PLGA and siRNA-PEI/(siRNA-DPPC/PLGA) before and af-ter nebulization. Scale bar is 0.2 lm. Field is representative of the sample.
Meanwhile, likely, as a consequence of the good aerosol-ization properties of PEI-containing formulations, 0.0137– 0.0032 and 0.0334– 0.0069 lg/cm2of hNPs were recovered from coverslips after nebulization of low and high concentra-tions of PEI/DPPC/PLGARhodhNPs, respectively.
Fate and cytotoxicity of aerosolized hNPs in TCCC
To study the cell uptake, fluorescently labeled hNPs were aerosolized on TCCC. After 24 hours postexposure to hNPs, TCCC were visualized by CLSM (Fig. 6). Results indicate that both DPPC/PLGARhodand PEI/DPPC/PLGARhodhNPs
(labeled in red) can effectively penetrate into cells and are localized intracellularly.
At the tested concentrations, the developed hNPs displayed no significant cytotoxicity compared to both sodium chloride and mannitol solutions aerosolized at the same concentrations of the samples ( p> 0.5) (Fig. 7A). On the other hand, a sig-nificant cytotoxic effect was observed when the cocultures were incubated with Triton-X as positive control ( p< 0.001 versus DPPC/PLGARhod and PEI/DPPC/PLGARhod). These
findings were confirmed by CLSM analysis of all treated
samples, since no alteration of cell morphology occurred after TCCC exposure to hNPs compared to the negative control (Supplementary Fig. S5).
The potential proinflammatory effect of aerosolized empty and siRNA-loaded hNPs on TCCC was also investigated by determining TNF-a secretion 24 hours postexposure (Fig. 7B). Of note, exposure to mannitol and empty hNPs, at both low and high concentration, caused a less than onefold increase of TNF-a releTNF-ase to the TCCC exposed to 0.5 mM TNF-aqueous sodium chloride. As for siRNA-loaded hNPs, we can see at all con-centrations tested a higher production of TNF-a in comparison to negative control, although values were not significantly different from the mannitol control.
FIG. 6. CLSM images of TCCC after 24 hours postexpo-sure to 0.5 mg of fluorescently labeled DPPC/PLGARhod(A)
and PEI/DPPC/PLGARhod(B) hNPs. Red color corresponds
to the hNPs, white color to cytoskeletal F-actin, and cyan color to nulcei. The arrows show hNPs engulfed by the cells. TCCC, triple cell coculture model. Color images available online at www.liebertpub.com/jamp
FIG. 7. LDH release (A) and TNF-a secretion (B) of TCCC after 24 hours postexposure to different theoretical amounts (0.1 and 0.5 mg) of aerosolized empty or siRNA-loaded hNPs. Sodium chloride was used as negative control. Mannitol aqueous solutions at the same volume and concentrations used for hNP samples were also aerosolized for comparison. Triton 0.2% (w/v) in PBS was used as positive control in LDH release (A). LDH data are presented as mean optical density (OD) values– SEM of samples diluted 1:10 v/v in cell culture medium (n= 3) (**p < 0.001). TNF-a data are presented as the mean fold increase of samples relative to the negative control (i.e., sodium chloride)– SEM (n = 3). LDH, lactate dehydrogenase; SEM, standard error mean; TNF-a, tumor necrosis factor alpha. Color images available online at www.liebertpub.com/jamp
In vitro silencing effect of siRNA-loaded hNPs
Once proven that the developed hNPs are able to overcome the lung barriers and reach the target cells, we further eval-uated the effect of siRNA-loaded hNPs on gene silencing efficiency in a human lung epithelial cell line, that is A549 cells. Preliminary cytotoxicity studies showed that both DPPC/PLGA and PEI/DPPC/PLGA hNPs exhibited a safe profile toward A549 cells in a wide range of concentrations (from 0.2 to 2 mg/mL) up to 72 hours of incubation (Sup-plementary Fig. S6). Nevertheless, to determine the amount of siRNA-loaded hNPs necessary to obtain an efficient gene de-pletion, dose–response studies were further performed by transiently transfecting A549 cells with increasing concentra-tions of siRNA pool by using lipofectamine. Results showed a specific and dose-dependent silencing of both aENaC and bENaC subunits (Supplementary Fig. S7). In particular, while nontargeting siRNA pool at 20 nM did not exert any effect on the levels of aENaC and bENaC subunits (data not shown), siRNA pool against ENaC resulted in robust protein knock-down at 24 hours (about 50% and 60% of gene depletion for aENaC and bENaC, respectively). This siRNA concentration was thus selected for in vitro gene silencing studies with
siRNA-loaded hNPs. We found that siRNA pool slowly re-leased from hNPs resulted in efficient knockdown of both aENaC and bENaC subunit proteins up to 72 hours (Fig. 8). Western blots confirmed that protein expression was signifi-cantly reduced by more than 60% and 40% for aENaC and bENaC, respectively, by siRNA-DPPC/PLGA hNPs compared to control (untreated cells). Of note, the addition of PEI inside the formulation increased the silencing efficiency of loaded hNPs by a further significant 10% compared to siRNA-DPPC/PLGA hNPs. The same amount of blank siRNA-DPPC/PLGA and PEI/DPPC/PLGA hNPs did not affect the levels of ENaC subunits (data not shown).
Discussion
The importance of lung barriers in determining the thera-peutic efficacy of inhaled drugs in severe lung diseases, such as CF, is nowadays acknowledged.(7)A current paradigm sug-gests that only NPs with appropriate size and surface properties may overcome the complex mucus airway barrier and assist drug interactions with human airway epithelial cells.(25,26)This aspect is particularly crucial for siRNA-based therapeutics, the clinical application of which is further hindered by the short half-life in vivo and the low capability to penetrate cell mem-branes. To overcome these issues, we have designed and de-veloped novel DPPC/PLGA hNPs, containing or not PEI as third component, suitable for pulmonary delivery of siRNA.
In line with literature data,(10)a DPPC layer is likely present on the surface of PLGA particles giving rise to a core-shell structure, which is independent of the presence of PEI. As demonstrated by hNP/mucus interaction studies, this structure results in non-mucoadhesive or muco-inert hNPs, reasonably able to easily penetrate across the airway mucus layer.(8,17) Furthermore, the adopted formulation conditions allowed the production of hNPs with a hydrodynamic diameter of 150 nm, which are expected to easily diffuse through the gel-like structure of the mucus.(25,27) Finally, aqueous hNPs were transformed into a solid long-term stable lyophilized powder, which can be reconstituted in saline and, likely, employed to co-administer a mucus rehydrating agent, that is mannitol.(24) If DPPC, which is the principal component of the lung lining fluids, is expected to tune hNP interactions with the lung environment, the addition of PEI can play a crucial role for siRNA entrapment inside DPPC/PLGA hNPs. This hy-pothesis was confirmed by encapsulation studies performed on two siRNA sequences, for a and b ENaC subunits, which have been shown to produce a long-term reduction of trans-epithelial Na+currents and fluid absorption.(28)In particular, the combination of the two siRNA can expand the lung fluid volume in vitro.(29) Of note, siRNA was entrapped inside selected hNPs with high efficiency, particularly when PEI was coencapsulated. This effect could be likely ascribed to the formation of a complex between PEI, employed at the theoretical N/P ratio of 10, and siRNA.
Information about the presence of PEI/siRNA complexes inside hNPs could not be derived from SAXS spectra, due to the very low amounts of both components. Nevertheless, SAXS analysis clearly demonstrated that the role of PEI in the formulation is not to affect the overall particle morphology, except for a very slight decrease of the average size. On the other hand, the phospholipid component is possibly coating the PLGA particles, as suggested by TEM analysis. Furthermore, FIG. 8. Representative Western blotting of protein extracts
from A549 cells treated or not with siRNA- DPPC/PLGA or siRNA-PEI/DPPC/PLGA for 72 hours. aENaC and bENaC proteins were detected by using the indicated antibodies. Anti-actin was used as the loading control (A). The quantification of signals is shown (B). The signal of untreated cells (NT) was set as 1.
independent of the presence of PEI inside the formulation, hNPs were stable inside artificial CF mucus at least for 12 hours, confirming, along with in vitro aerosol performance studies, the potential of the system for direct aerosolization on the mucus-lined human airway epithelial barrier.
In fact, to attain maximum siRNA availability at lung epithelial cells, where its target is located, the hNPs should (i) uniformly deposit on the airway epithelium in their stable form; (ii) penetrate inside the lung epithelial barrier; and (iii) interact with the lung epithelium, without compromis-ing cell availability. To draw key information on all these points and to establish a priori target formulation parameters for siRNA delivery to human airway epithelium, the be-havior of the developed DPPC/PLGA hNPs after deposition on triple cell co-cultures, which realistically mimic human airway epithelium environment, was investigated.
When grown on porous supports at ALI, human bronchial epithelial cells reproduce in vivo morphology and key phys-iologic processes, sometimes including tight junctions and basal and mucus cells.(19,30)Furthermore, direct deposition of the aerosolized drug to the apical compartment of the cell culture can be envisaged through systems especially designed for dose-controlled and spatially uniform deposition of liquid aerosols on cells,(31,32)such as the Vitrocell Cloud employed in this study. Results showed that the adopted formulation conditions allowed to tune hNP properties, so as to achieve uniform and efficient aerosolization on lung epithelial cells in their integer form. Afterward, hNPs were demonstrated to penetrate inside extracellular lung lining fluids, to be taken up by lung epithelial cells without exerting a cytotoxic effect or a proinflammatory response on the treated lung epithe-lium model.
Once siRNA molecules overcome the aforementioned lung epithelial barriers and reach the target cells, they still need to overcome a series of intracellular barriers to exert their ac-tivity. Following cell entry, siRNA molecules must escape early from endosomes to avoid exposure to the noxious acidic environment as well as late-stage attack by the hydrolases in the endolysosome.(4) Noteworthy, in vitro ENaC silencing studies ruled out any degradation process, while proving the ability of the siRNA released from hNPs to reach the cyto-plasm and, after binding to its target mRNA, to silence the gene of interest. As a consequence, robust and long-lasting knockdown of both aENaC and bENaC proteins could be achieved.
Conclusions
In this work, we have successfully designed and developed novel hNPs comprising PLGA likely surrounded by a DPPC shell, showing size and surface properties optimized for siRNA transport and delivery at lung. Optimized formulations allowed the encapsulation of a combination of siRNA against a and b subunits of ENaC channel with high efficiency, which could be optimized by the addition of PEI inside the formulation. The developed hNPs were muco-inert and stable inside AM. Stable hNP-based dry powders were produced by freeze-drying with mannitol, an osmotic agent of interest for CF treatment. Cell uptake studies upon in vitro aerosolization on TCCC con-firmed the ability of fluorescently labeled hNPs to be inter-nalized inside the airway epithelial barrier. Hazard assessment of hNPs demonstrated that the developed system does not exert
cytotoxic or acute proinflammatory effect toward any of the cell components of the co-culture model. Finally, in vitro gene silencing studies highlighted that released siRNAs are capable to reduce ENaC protein expression in human lung epithelial cells. Overall, results demonstrate the great potential of DPPC/ PLGA hNPs as carriers for siRNA delivery on the human ep-ithelial airway barrier, prompting toward investigation of their therapeutic effectiveness in severe lung diseases.
Acknowledgments
F.U. gratefully acknowledges Compagnia di San Paolo and Istituto Banco di Napoli—Fondazione (STAR Program, Ju-nior Principal Investigator Grant 2013, Napoli_call2013_35) for financial support. G.C. wishes to thank the International Society for Aerosols in Medicine (ISAM) for the 2015 ISAM Student Fellowships. E.D., A.P.F., and B.R.R. acknowledge the financial support of the Adolphe Merkle Foundation. The authors are grateful to Dr. T. Nrayanan, Dr. J. Moller, and to the ID02 beamline staff at the European Synchrotron Facility (Grenoble, France) and to Dr. Laetitia Haenni and Dr. Christoph Monnier at TEM facility of the Adolphe Merkle Institute for precious technical assistance.
Author Disclosure Statement
No competing financial interests exist.
References
1. Burnett JC, Rossi JJ, and Tiemann K: Current progress of siRNA/shRNA therapeutics in clinical trials. Biotechnol J. 2011;6:1130–1146.
2. Lee SH, Kang YY, Jang HE, and Mok H: Current pre-clinical small interfering RNA (siRNA)-based conjugate systems for RNA therapeutics. Adv Drug Deliv Rev. 2016; 104:78–92.
3. Karras JG, Sun G, Tay J, and Jackson AL: Reflections on microRNAs in chronic pulmonary disease: Looking into the miR-ror and crystal ball. Inflamm Allergy Drug Targets. 2013;12:88–98.
4. Lam JK, Liang W, and Chan HK: Pulmonary delivery of therapeutic siRNA. Adv Drug Deliv Rev. 2012;64:1–15. 5. Merkel OM, Rubinstein I, and Kissel T: siRNA delivery to the
lung: What’s new? Adv Drug Deliv Rev. 2014;75:112–128. 6. Ungaro F, d’Angelo I, Miro A, La Rotonda MI, and
Qua-glia F: Engineered PLGA nano- and micro-carriers for pulmonary delivery: Challenges and promises. J.Pharm Pharmacol. 2012;64:1217–1235.
7. d’Angelo I, Conte C, La Rotonda MI, Miro A, Quaglia F, and Ungaro F: Improving the efficacy of inhaled drugs in cystic fibrosis: Challenges and emerging drug delivery strategies. Adv Drug Deliv Rev. 2014;75:92–111.
8. d’Angelo I, Casciaro B, Miro A, Quaglia F, Mangoni ML, and Ungaro F: Overcoming barriers in Pseudomonas aer-uginosa lung infections: Engineered nanoparticles for local delivery of a cationic antimicrobial peptide. Colloids Surf B Biointerfaces. 2015;135:717–725.
9. Griffiths PC, Cattoz B, Ibrahim MS, and Anuonye JC: Probing the interaction of nanoparticles with mucin for drug delivery applications using dynamic light scattering. Eur J Pharm Biopharm. 2015;97:218–222.
10. Raemdonck K, Braeckmans K, Demeester J, and De Smedt SC: Merging the best of both worlds: Hybrid lipid-enveloped
matrix nanocomposites in drug delivery. Chem Soc Rev. 2014;43:444–472.
11. Pandita D, Kumar S, and Lather V: Hybrid poly(lactic-co-glycolic acid) nanoparticles: Design and delivery prospec-tives. Drug Discov Today 2015;20:95–104.
12. Ungaro F, Giovino C, Coletta C, Sorrentino R, Miro A, and Quaglia F: Engineering gas-foamed large porous particles for efficient local delivery of macromolecules to the lung. Eur J Pharm Sci. 2010;41:60–70.
13. De Stefano D, Ungaro F, Giovino C, Polimeno A, Quaglia F, and Carnuccio R: Sustained inhibition of IL-6 and IL-8 expression by decoy ODN to NF-kappaB delivered through respirable large porous particles in LPS-stimulated cystic fibrosis bronchial cells. J Gene Med. 2011;13:200– 208.
14. Blank F, Rothen-Rutishauser B, and Gehr P: Dendritic cells and macrophages form a transepithelial network against for-eign particulate antigens. Am J Respir Cell Mol Biol. 2007; 36:669–677.
15. Maiolino S, Moret F, Conte C, Fraix A, Tirino P, Ungaro F, Sortino S, Reddi E, and Quaglia F: Hyaluronan-decorated polymer nanoparticles targeting the CD44 receptor for the combined photo/chemo-therapy of cancer. Nanoscale. 2015; 7:5643–5653.
16. Narayanan T, Diat O, and B+Aˆ secke P: SAXS and USAXS
on the high brilliance beamline at the ESRF. Nuclear In-struments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment 2001;467–468, Part 2:1005–1009.
17. Ungaro F, d’Angelo I, Coletta C, d’Emmanuele di Villa Bianca R, Sorrentino R, Perfetto B, Tufano MA, Miro A, La Rotonda MI, and Quaglia F: Dry powders based on PLGA nanoparticles for pulmonary delivery of antibiotics: Modulation of encapsulation efficiency, release rate and lung deposition pattern by hydrophilic polymers. J Control Release. 2012;157:149–159.
18. Marple VA, Olson BA, Santhanakrishnan K, Roberts DL, Mitchell JP, and Hudson-Curtis BL: Next generation pharmaceutical impactor: A new impactor for pharmaceu-tical inhaler testing. Part III. extension of archival cali-bration to 15 L/min. J Aerosol Med. 2004;17:335–343. 19. Lehmann AD, Daum N, Bur M, Lehr CM, Gehr P, and
Rothen-Rutishauser BM: An in vitro triple cell coculture model with primary cells mimicking the human alveolar ep-ithelial barrier. Eur J Pharm Biopharm. 2011;77:398–406. 20a. Russo A, Saide A, Cagliani R, Cantile M, Botti G, and
Russo G: rpL3 promotes the apoptosis of p53 mutated lung cancer cells by down-regulating CBS and NFjB upon 5-FU treatment. Sci Rep. 2016;6:38369.
20b. Pagliara V, Saide A, Mitidieri E, d’Emmanuele di Villa Bianca R, Sorrentino R, Russo G, and Russo A: 5-FU targets rpL3 to induce mitochondrial apoptosis via cystathionine-b-synthase in colon cancer cells lacking p53. Oncotarget. 2016;7:50333–50348.
21. De Filippis D, Russo A, De SD, Cipriano M, Esposito D, Grassia G, Carnuccio R, Russo G, and Iuvone T: Palmi-toylethanolamide inhibits rMCP-5 expression by regulating MITF activation in rat chronic granulomatous inflamma-tion. Eur J Pharmacol. 2014;725:64–69.
22. Guinier G, and Fournet G: Small Angle Scattering of X-Rays. John Wiley & sons, New York, 1955. pp. 126–160. 23. Fonte P, Reis S, and Sarmento B: Facts and evidences on
the lyophilization of polymeric nanoparticles for drug de-livery. J Control Release 2016;225:75–86.
24. Nolan SJ, Thornton J, Murray CS, and Dwyer T: Inhaled mannitol for cystic fibrosis. Cochrane Database Syst Rev. 2015;10:CD008649.
25. Ensign LM, Tang BC, Wang YY, Tse TA, Hoen T, Cone R, and Hanes J: Mucus-penetrating nanoparticles for vaginal drug delivery protect against herpes simplex virus. Sci Transl Med. 2012;4:138–179.
26. d’Angelo I, Conte C, Miro A, Quaglia F, and Ungaro F: Pulmonary drug delivery: A role for polymeric nano-particles? Curr Top Med Chem. 2015;15:386–400. 27. Lai SK, Wang YY, Hida K, Cone R, and Hanes J:
Nano-particles reveal that human cervicovaginal mucus is riddled with pores larger than viruses. Proc Natl Acad Sci U S A. 2010;107:598–603.
28. Caci E, Melani R, Pedemonte N, Yueksekdag G, Ravazzolo R, Rosenecker J, Galietta LJ, and Zegarra-Moran O: Epi-thelial sodium channel inhibition in primary human bron-chial epithelia by transfected siRNA. Am J Respir Cell Mol Biol. 2009;40:211–216.
29. Gianotti A, Melani R, Caci E, Sondo E, Ravazzolo R, Galietta LJ, and Zegarra-Moran O: Epithelial sodium channel silencing as a strategy to correct the airway surface fluid deficit in cystic fibrosis. Am J Respir Cell Mol Biol. 2013;49:445–452.
30. Rothen-Rutishauser BM, Kiama SG, and Gehr P: A three-dimensional cellular model of the human respiratory tract to study the interaction with particles. Am J Respir Cell Mol Biol. 2005;32:281–289.
31. Endes C, Schmid O, Kinnear C, Mueller S, Camarero-Espinosa S, Vanhecke D, Foster EJ, Petri-Fink A, Rothen-Rutishauser B, Weder C, and Clift MJ: An in vitro testing strategy toward mimicking the inhalation of high aspect ratio nanoparticles. Part FibreToxicol. 2014;11:40. 32. Endes C, Mueller S, Kinnear C, Vanhecke D, Foster EJ,
Petri-Fink A, Weder C, Clift MJ, and Rothen-Rutishauser B: Fate of cellulose nanocrystal aerosols deposited on the lung cell sur-face in vitro. Biomacromolecules. 2015;16:1267–1275.
Received on January 3, 2017 in final form, July 26, 2017
Reviewed by: Olivia Merkel Sally-Ann Cryan
Address correspondence to: Barbara Rothen-Rutishauser, PhD Adolphe Merkle Institute University of Fribourg Chemin des Verdiers 4 Fribourg 1700 Switzerland E-mail: [email protected]
Francesca Ungaro, PhD Department of Pharmacy University of Napoli Federico II Via D. Montesano 49 Napoli 80131 Italy E-mail: [email protected]
